Chemotaxis by the RAW264 mouse macrophage cell line was inhibited by 3-deazaadenosine but not by 3-deazaaristeromycin. Observations of cells by time-lapse video photography indicated that treatment of cells with 3-deazaadenoside inhibited signal processing. A search for biochemical reactions inhibited by 3-deazaadenosine but not by 3-deazaaristeromycin has revealed that only one reactions, the synthesis of a small number of proteins identified after separation by two-dimensional polyacrylamide gel electrophoresis, has the necessary inhibitor specificity for involvement in the 3-deazaadenosine-sensitive step of chemotaxis. A correlation was found between inhibition of chemotaxis and inhibition of the synthesis of same subset of proteins when other compounds were tested. These compounds also inhibited the synthesis of polyadenylated mRNA, leading us to postulate that incubation of cells with 3-deazaadenosine inhibits a methylation reaction that is required for the formation of functional mRNA coding for one or more proteins required for chemotaxis. Experiments with broken cells indicated that the inhibition of the synthesis of proteins by 3-deazaadenosine was not at the level of translation. Experiments to identify attractant-specific proteins have been limited because chemically defined attractants for RAW264 cells have not been available. This problem has been overcome by the isolation of a stable cell hybrid from a fusion between human leukocytes and a thioguanine-resistant RAW264 cell line. The hybrid expressed functional genes for chemotaxis to N-formylmet-leu-phe, a commercially available synthetic attractant. The hybrid was similar to RAW264 cells in morphology and inhibition by 3-deazaadenosine and 3-deazaaristeromycin. Bacterial toxins may provide another probe of reactions involved in chemotaxis. RAW264 chemotaxis was inhibited by cholera toxin and pertussis toxin. Cholera toxin did not appear to exert its inhibitory action by increasing the levels of cAMP, since treatment of RAW264 cells with other compounds which increased cAMP to comparable levels, such as forskolin and isoproterenol, did not inhibit chemotaxis.